Project 2 ? Replication Fork Repair PROJECT SUMMARY/ABSTRACT Despite active repair and proofreading mechanisms, the replication machinery encounters unrepaired lesions and other forms of replication stress every cell division cycle. Therefore, completing DNA replication faithfully requires specialized replication stress response and error correction mechanisms. Replication fork remodeling by DNA translocases and nucleases can stabilize and repair damaged replication forks while mismatch repair enzymes can correct polymerase errors. However, inactivation or improper regulation of these enzymes generates DNA sequence changes that fuel cancer development. For example, inactivation of mismatch repair (MMR) is the most frequent cause of inherited cancers. Furthermore, oncogenes generate elevated levels of replication stress. While the genetic instability that results from these changes can promote tumorigenesis, it also makes cancer cells more dependent on the remaining replication stress response and repair pathways. Thus, these properties of cancer cells provide therapeutic opportunities that can be exploited by both traditional chemotherapeutic and radiation therapies that target DNA and newer agents like PARP inhibitors that more selectively utilize synthetic lethality to kill cancer cells. The guiding principle of this project is that understanding how replication stress and fork repair activities work in normal and cancer cells is critical to understand both the etiology of cancer and to develop and deploy new therapies. A five-member team of investigators with expertise spanning structural biology, biochemistry, biophysics, genetics, and cell biology will focus on the key regulatory nodes that direct replication-associated repair activities. We will capitalize on the progress made in the last funding period, the ongoing research in project member laboratories, and the synergy created by employing multiple experimental approaches to address the following specific aims: (1) define the mechanisms by which the fork remodeling proteins ZRANB3, HLTF, and SMARCAL1 repair damaged replication forks; (2) define the unique replication-associated cellular functions of fork remodeling proteins; and (3) define mechanisms controlling nuclease activities at replication forks. Collaborations with other SBDR projects will ensure the highest impact of our studies. For example, we will work with project 1 to understand RPA function, project 3 to examine activities of HRR proteins in fork repair, project 4 to explore the mechanism of action of PARP inhibitors, and project 5 to examine the role of MRN proteins at replication forks.